The major reserve carbohydrates found in vascular plants are sucrose, starch and fructan, (a non-reducing polymer of fructose linked to a terminal glucose residue). Despite numerous agronomic and technical barriers, crops are grown throughout the world specifically as sources of sucrose or starch for use primarily, in the sweetener industry.
Economically successful cultivation and processing of sugar beet and sugarcane for sucrose must overcome obstacles including, but not limited to, restricted growing regions, labor intensive harvesting practices, critical timing of harvest when sucrose levels reach their peak, undesirable changes in composition and quality brought on by delay in transport or processing, and yield loss due to improper or long term storage (Salunkhe and Desai, Postharvest Technol. of Sugar Crops, CRC Press, Boca Raton, Fla. (1988); Stout, J. Am. Soc. Sugar Beet Technol., 9:350 (1957); Barnes, The Sugarcane, 2nd. ed., Leonard Hill Books, London (1974)). Processing of sugarcane for example, only 9 days after it has been cut is an unprofitable exercise due to the tremendous loss of sucrose by enzymatic degradation, (Alexander, Sugarcane Physiology, Elsvier, Amsterdam, (1973); Gulibeau et al., Sugar J., 18:30 (1955)). Because of the relatively short period of profitability, the timing of harvest and processing requires rigorous planning. Unexpected delays, such as those for extreme weather conditions, may result in significant loss of product. Optimum harvest periods for sugar beet are also complicated by issues of timing. Raffinose, the primary contaminant of beet juice, inhibits the crystallization of sucrose and presents a considerable challenge to profitable sugar beet processing. Raffinose has been shown to increase dramatically during the same period when sucrose levels peak and continues to increase during storage of beets at temperatures needed to prevent sucrose degradation (Finkler et al., J. Am. Soc. Sugar Beet Technol., 10:459 (1959); Brown, Anal. Chem., 24:384 (1952)). Cultivation of sugar beet for sucrose is complicated then, because the level of sucrose and amount of raffinose determine the quality and therefore the profitability of sugar beet processing.
Starch based sweeteners, produced mostly from corn, were developed in part because of the many limitations associated with sugarcane and sugar beets. Corn sweeteners also helped to relieve dependence on importation of sucrose. Supply had historically, been subject to world shortages and volatile price swings, brought about through a number of political events and natural diastase.
The shift away from sucrose crops in the United States was remarkably rapid, possibly due to the many advantages inherent in producing sweeteners from starch. One, for example is that harvest and storage conditions for corn are much more favorable compared to sucrose crops such as sugar beet and sugarcane. This allows much longer storage time, without quality loss while waiting for available process capacity. Profitable production of sucrose from sugarcane must take place within a few days of harvest to prevent quality losses. In contrast, corn may be held in proper storage for a year before isolation of starch for sweetener production, without significant loss or alteration of product. Another advantage corn has over crops such as sugarcane, is the adaptation to a greater variety of growing conditions. The use of corn vastly increased the amount of acres in the United States available to growing a crop for the sweetener industry. Furthermore, fructose, the end product of starch based sweeteners, is preferred over sucrose by major consumers due to its enhanced relative sweetness. Under acidic conditions, fructose may be up to 1.8 times sweeter than sucrose. This results in a savings to the consumer because less product may be used to produce the same effect.
Despite the commercial success of the fructose corn syrup industry, there is considerable room for improvement. Technical hurdles that must be overcome to convert starch, a glucose polymer, into fructose add significantly to the cost of production. The current technology of fructose syrup production essentially begins with the hydrolysis of starch into individual glucose residues. This is accomplished by enzymatic hydrolysis, in order to reduce off colors, flavors and poor yield due to unusable oligomers formed when hydrolysis of starch is accomplished through a mild acid treatment. Enzymatic hydrolysis of starch, efficient though it may be, results in significant monetary loss because of the massive scale of fructose syrup production. A 96% conversion of starch to glucose results in a tremendous loss in potential product, considering the several hundred million pounds of starch hydrolyzed each year. With as little as 4% loss, due to incomplete hydrolysis of starch, the result may be a loss amounting to tens of millions of pounds of potential glucose that would otherwise be sold as the final product. The effect is a loss of millions of dollars of potential revenue each year.
The efficiency of the total process is reduced even further during the isomerization step. Conversion of glucose to fructose, enzymatically, reaches equilibrium at only 42% fructose. This mixture of fructose and glucose is not an acceptable product. The industry standard is sucrose and the 42% fructose solution is not comparable in terms of sweetness to an equivalent sucrose solution. The consequence is that the 42% solution must be enriched for fructose by ion exchange chromatography which adds significantly to the total cost of production. A 90% fructose solution is eluted from the ion exchange column and blended with a portion of the 42% fructose solution to obtain a 55% product. The 55% fructose solution has an equivalent level of sweetness compared to sucrose.
The fastest growing market for fructose sweeteners today is for crystalline fructose. This product is used in a growing number of baked goods and dry mixes. Production of crystalline fructose requires that the 90% fructose syrup isolated from ion exchange columns undergo additional processing, which adds to its cost. Additional processing is necessary to further remove glucose contamination before crystallization of fructose is possible. A mixture of approximately 97% fructose is needed to obtain this product which sells for a premium.
Conversion of a glucose polymer (starch), into fructose has a number of advantages in the sweetener industry, but they come at significant cost. The issue of cost could be addressed by substituting fructan as the starting material for fructose syrup production. The third member of plant carbohydrate storage reserves fructans, have been known for over 150 years. Fructans consist of individual fructose residues connected by .beta.2-1 and .beta.2-6 linkages. Simple hydrolysis, of this polymer by either enzymatic or mild acid treatment yields substantially pure fructose. Fructans, therefore, offer a unique advantage over starch in purity and content that would result in the elimination of saccharification, isomerization, and ion exchange steps, currently utilized in fructose syrup production. Simplified processing, resulting in a reduction of costs and the higher relative sweetness of fructose compared to sucrose are only two reasons why fructans are considered to be excellent starting materials for the sweetener industry.
The disadvantages of cultivating fructan containing crops however, are comparable to those grown for sucrose. Although 23 separate plant families have been shown to accumulate fructans (Hendry, New Phytol., 106:201-216 (1987); Nelson and Spollen, Physiol. Plant, 71:512-516 (1987)), only two species are considered as potential industrial crops. Jerusalem artichoke (Helianthus tuberosus) and Chicory (Cichorium intybus), are known to be as productive as traditional agricultural crops, accumulating carbohydrate levels, comparable to sugar beet and potato (Fuchs, Starch 39:335-43 (1987)). Jerusalem artichoke and chicory are however, seasonable crops. Fructans are stored in below ground tubers during only a portion of the growing season. Fructan synthesis falls off rapidly after tuber development ceases. Degradation activity increases in the tuber during maturation and remains high during the dormant period (Fuchs, Starch 39:335-43 (1987)). Timing of harvest is particularly critical for Jerusalem artichoke. The desirable larger molecular weight polymers are more prevalent in young tubers when relatively little of the potential amount of fructan has accumulated. The mature tubers contain up to 80% of their dry weight as fructan, but the predominant species are of the lower molecular weight type, (Haunold et al., J. Plant Physiol. 134:218-223 (1989)). Because all fructans contain a terminal glucose residue, originating from the starting sucrose molecule, the larger the number of fructose residues the more pure the polymer. Less purification of fructose from glucose contamination is needed then, with the larger fructans. Intensive processing of fructans during a short, but critical time of the year may circumvent losses due to degradation, but this would be subject to processing capacity. Processing capacity would in turn, certainly be limited by the costs needed to build specialized equipment and commercial plants capable of processing a unique root crop, during the short harvest season.
A major disadvantage of cultivating Jerusalem artichoke for commercial harvest of fructan is the thin delicate skin of the tuber. Injury to the tuber often occurs during harvest, increasing respiration, which results in high water loss and increased fructan degradation during storage. Storage under ambient conditions is limited to only a few weeks, at best before significant degradation of fructans reduces economic success. Alternatively, the crop could be maintained in storage at a constant temperature and humidity until processed. This would prevent product loss (Dykins et al., Industrial and Engineering Chemistry, 25:937-940 (1933)) however, the expense involved for large scale storage is prohibitive.
Fructan accumulation in the field is extremely sensitive to environmental change. Exposure to drought or frost dramatically alters the quality of the fructan accumulated (Praznik and Beck, Agr. Biol. Chem., 51:1593-1599 (1987)). Traditional breeding programs could in theory, result in varieties with reduced quality losses due to environmental change. However, programs of this type, normally very time consuming, are not in place at this time and would likely be implemented only when the fructan industry proves to be viable. Genetic engineering of fructan containing crops could also eliminate these barriers. Overexpression of a fructan biosynthetic gene or genes, may lead to increased yield, synthesis of larger molecular weight fructans or reduced quality losses due to frost or drought. This approach could also potentially eliminate the need for specialized storage conditions. Success of such a genetic program would rely heavily on a detailed understanding of the biochemistry of fructan synthesis, the kinetics of the biosynthetic proteins and ultimately, understanding the regulation of the genes involved in fructan synthesis. At present, this knowledge is lacking. The current model for all fructan accumulating plants, proposed in 1968 (Eddleman and Jefford, New Phytol. 67:517-531 (1968)), suggests that polymer synthesis and storage is achieved by the sequential action of two separate proteins. The model, which has been slightly altered, (Wagner et al., Zeitschrigt fur Pflanzenphysiologie 112:359-372, (1983); Frehner et al. 1984, New Phytol. 116:197-208) has yet to resolve a key issue regarding reversible fructosyltransferase (FTF) activity and is once again under critical review (Housley et al., New Phytol. 119:491-97, (1991); Cairns, A. J., New Phytol. 120:463-73 (1992)). Enzymes involved in the biosynthesis pathway have not been purified to homogeneity. Therefore, attempts to fully understand fructan metabolism and then to alter regulation of synthesis, control loss due to degradation and increase the molecular weight of accumulated fructans in a transgenic plant using a cloned plant gene or genes may be several years away.
Microorganisms are also known to produce fructans. However, unlike plant systems, microbial fructan synthesis is well characterized (reviewed in: Hehre, Adv. in Enzymol., 11:297 (1951); Hestrin, The Bacteria: A Treatise on Structure and Function, Academic Press, NY, Gunsalas and Stanier eds., Vol. 3, chap. 8 (1962)) FTFs derived from bacterial sources catalyze the polymerization of linear or branched polymers containing .beta.2-1, .beta.2-6 or combinations of .beta.2-1 and .beta.2-6 linked fructose residues. Chains of fructan, similar to starch and dextran, grow by a step-by-step addition of a single fructofuranosyl residue at the C-6 hydroxyl of the nonreducing fructose terminal unit in the growing chain. Alternatively, branches in the chain occur when the addition of fructose residues occurs at the C-1 hydroxyl. Branching may occur at a rate of up to 12% of the polymer (Hestrin, Ann. N.Y. Acad. Sci., 66:401 (1956); Hehre, Methods Enzymol. 1:178-192 (1955); Han, Adv. Appl. Microbiol. 35:171-194 (1990)). Most extensively studied in Bacillus subtilis, many species have been identified that posses fructose polymerizing activity (Evans and Hibbert, Adv. Carbohydr. Chem., 2:253-277 (1946); Mantsala and Puntala, FEMS Microbio. Lett., 13:395-399 (1982); Kleczowski and Wierzchowski, Soil Sci., 49:193 (1940)). Bacterial proteins have been purified to homogeneity (Chambert et al. Eur. J. Biochem. 41:285-300 (1974)) and crystallized (Berthou et al., J. Mol. Biol., 82:111-13 (1974)). Exhaustive study of the purified bacterial FTF activity led to the finding that polymers are synthesized by a single protein acting on sucrose, the sole substrate. The fructose chain grows by the repeated transfer of fructose from a donor sucrose to an acceptor fructan polymer. Synthesis has been demonstrated to be independent of the need for cofactors or primers. Purified protein allowed identification and cloning of the bacterial FTF gene from several species (Fouet, A., Arnaud, M., Klier, A. and Rapoport, G., Biochem. Biophys. Res. Commun. 119, 795-800 (1984); Shiroza, T. and Kuramitsu, H. K., J. Bacteriol. 170, 810-816 (1988); Tang, L. B., Lenstra, R., Borchert, T. V. and Nagarajan, V. Gene 96, 89-93 (1990)). The cloned genes and site directed mutagenesis provided additional information concerning binding regions, kinetics and intermediate protein-sugar complexes (Chambert, R., and Petit-Glatron, M. F., Biochem. J., 279, 35-41 (1991)).
Microbial fructan biosynthesis is well understood allowing regulation of plant fructan accumulation through genetic engineering. The cloned bacterial genes present opportunities to alter fructan containing crop species, or to accumulate fructans in transgenic agricultural crops where they are not normally found in nature. However, with sucrose as the sole substrate, many potential barriers to successful expression in a transgenic plant must be considered. Expression of a bacterial gene with sucrose metabolic properties in a transgenic plant must be in consideration of the critical role sucrose plays in the growth and development of higher plants. Most compounds formed in nonphotosynthetic tissues of a plant are derived from sucrose. Sucrose concentration has been shown to regulate gene expression (Visser et al., Plant Mol. Biol., 17:691-699 (1991); Wentzler et al., Plant Mol. Biol., 12:41-50 (1989)) and has a demonstrated role in regulating the rate of photosynthesis, (Stitt et al., Planta, 183:40-50 (1990); Krapp et al., The Plant J., 3:817-828 (1993)). These roles cannot, and should be ignored. Indiscriminate expression of a gene with the capacity to alter sucrose concentration in a transgenic plant may deprive nonphotosynthetic tissue of a crucial metabolite where it is most needed and could have serious consequences on the development of that tissue. Altered concentration would also alter gene expression, linked to sucrose level in the cell, leading to unknown, but certainly serious negative results.
Specialized structures in higher plants exist to transfer, collect and concentrate sucrose. Sucrose levels therefore, are considerably varied throughout a plant, within cellular organelles and among species. Although sucrose is the dominant form of carbohydrate transported from net carbon exporting tissue (source) to net carbon importing tissue (sink), many plants transport alternate forms of sucrose (e.g., raffinose) or alternate carbohydrates altogether (e.g., mannitol or sorbitol). Successful expression of a sucrose metabolizing enzyme across a varied population of plant species, without altering regulatory signals and subcellular expression sequences is then, highly unlikely. Expression must only be in consideration of the multiple mechanisms that exist to transport and concentrate sucrose, altered forms of sucrose that exist in higher plants and the critical role sucrose plays in various plant tissues.
Accumulation of bacterial fructans in transgenic plants offers several advantages over plant fructans. Fructan size is the most notable difference between fructans from plant and microbial sources. Plant polymers are low molecular weight with an average of 10-30 fructose units per molecule. In contrast, microbial fructans may contain over 100,000 fructose residues with a molecular weight of up to 10.sup.6 -10.sup.8. Increased fructan size, in the context of this invention, is a great advantage because the larger the polymer, the greater the fructose to glucose ratio and the less purification necessary to remove contaminating glucose following hydrolysis. Increased size is also an advantage because the larger bacterial fructans are much less water soluble than are the smaller plant polymers. The difference in solubility may be taken advantage of when processing tissue. Separating fructans from highly soluble cell material such as sucrose, glucose and other sugars, would be less technically difficult if the polymers to be isolated, were of the larger size. The large fructans also offer the opportunity to store more fructose in a cell without altering internal osmotic pressure compared to the same amount of fructose in smaller polymers. Since altering osmotic pressure in a sink tissue is critical to import of carbon, this advantage may be most significant of all.
Fructan accumulation in a transgenic plant may be an attractive alternative to the current fructose sweetener technology. Especially true in corn, a fructose polymer will not alter the advantages gained over sucrose crops, but instead, builds on them. Fructan production in corn for example, allows the utilization of the corn by-products (oil, meal and gluten feed) in addition to removing the tremendous costs of converting glucose to fructose. Hydrolysis of fructan into individual fructose residues results in a product consisting of at least 99% fructose. This highly pure product provides an alternative to the inefficient isomerization step and eliminates the need for fructose enrichment by ion exchange chromatography. Crystallization of fructose is simplified by starting with material that consists of 99%(+) fructose.
Reducing the cost of production is significant not only to the sweetener industry, but the use of fructose as a chemical feedstock is dependent on availability, purity and competitive price. At present the fructose industry can meet only the demands of purity. The United States is the largest producer of fructose syrups but, is a net importer of fructose. Food uses currently consume more fructose than is produced. Availability at a competitive cost would allow fructose, easily dehydrated to 5-hydroxymethyl-furfural (HMF) to be utilized as a building block for pharmaceuticals, such as Ranitidine or Zantac.TM., currently the best selling antiulcer drug. HMF may also be used as starting material for polymers, such as Kevlar.TM., and Nomex.TM., in addition to the potential for use in opto-electronic devices, due to the special optical effects of the furan nucleus (Schiweck et al., in Carbohydrates as Organic Raw Materials, Lichtenthaler ed., VCH Press, NY, pp. 72-82, (1992)). HMF may be converted into carbocyclic and heterocyclic compounds, creating a role in almost every part of applied chemistry, if only its purity could be combined with increased production and reduced cost.
The addition of very low levels of fructans in feed preparations was recently shown to bring about several positive metabolic and physiological changes in monogastric animals (Hashimoto et al., U.S. Pat. No. 4,734,402 (1988); Nakamura et al., U.S. Pat. No. 4,788,065 (1988); Farnworth et al., Inulin and Inulin Containing Crops, Fuchs ed., Elsvier, Amsterdam, pp 385-389 (1993)). The probiotic effect of fructans in feed may be attributed to an increase in the population of beneficial microflora in the intestine. Reduced instances of scours and increased feed efficiency has obvious potential benefit for domestic animal production. Accumulation of fructan within the grain is an advantage not only for the value as a probiotic, but allows "on farm" use without the need for expensive equipment needed in grinding or blending feeds.
Transformation of plants with a FTF results in the introduction of a gene that would not otherwise be possible through traditional breeding, but would take advantage of inbred or elite lines, well adapted to specific growing regions. Transforming with a bacterial FTF gene will result in a renewable source of a valuable polymer without the loss of established co-products such as oil, gluten feed and meal, in the case of corn. Transgenics also offer the advantage of accumulation of a fructan in a plant that does not have the capability of degrading that polymer. This means that environmental changes will not alter quality or quantity of the polymer as seen in plants such as Jerusalem artichoke and chicory. The transgenic tissue could be stored with less concern for degradation. Long term storage in unspecialized containers will reduce or eliminate the costs and technical needs associated with harvest and isolation from current fructan crops.
Methods described in this invention would enable commercial scale production of fructose polymers, as well as polymers of glucose. FTFs belong to a group of similar proteins known as sucrases which posses the ability to polymerize carbohydrate, using sucrose as the sole substrate. The sucrase family of proteins are similar in many respects for example, the proteins are catalytically active as monomers and no cofactors or primers are required for synthesis. The family of proteins would be expected to function as does the chimeric FTF in transgenic plants, based on the remarkable similarities within the group. The final product may contain polymerized fructose as is catalyzed by FTFs, but glucose may also be polymerized by sucrases known as glucosyltransferases (GTFs). GTFs vary in source as well as in function, and the type of polymer catalyzed varies accordingly. A number of GTFs (i.e., alternansucrase, GTF-I, GTF-S and GTF-SI, (Cote, Carbo. Polym., 19:249-252 (1992); Giffard et al., J. Gen. Micro., 139:1511-1522 (1993)) have been identified and each catalyzes the formation of a slightly different polymer. The polymer may vary in size, in linkage type or in pattern of linkages. As is true with starch, also a glucose polymer, the difference in size, linkage type and pattern of linkages determines the properties, which influences its commercial use. GTFs, such as certain dextransucrases, may polymerize glucose through unique linkages, resulting in properties very different from those of starch. GTFs are currently used to produce a glucose polymer, dextran for high value uses in research and as a volume extender of blood plasma. Large scale production of these alternate polymers offers options very much like those described for fructans, including providing a renewable source of unique polymers, reducing the production cost of polymers with demonstrated markets and opening markets through uses that would not otherwise be cost-effective.
WO89/12386 describes a method for the production of glucose and fructose polymers in transgenic tomato plants. The disclosure in that patent application describes exposure in the cytosol which may not be enabling, and further, results in destruction of transformed cells. WO89/12386 does not teach insertion in the vacuole.
The present invention details a method and the materials necessary for the synthesis and accumulation of fructose polymers in a transgenic plant where the polymer does not normally exist and in plant species without the ability to hydrolyze or alter the qualities of the polymer once accumulated. Accumulation of fructose polymers in transgenic plants has been accomplished through the tissue specific and sub-cellular expression of a bacterial fructosyltransferase (FTF) gene, using sucrose as a sole substrate and requiring no cofactors or externally supplied primers. Particular attention is paid to the level of sucrose in a particular cell, the timing of FTF expression, tissue specific expression in plant species and to subcellular location of expression. These issues, critical to the success of the invention, were not described or considered in previous publications.
This present invention describes a unique combination of tissue specific promoters, a vacuole targeting sequence, a coding sequence for a microbial FTF and a method for transferring the DNA fragments into tobacco, potato and corn. The result is a method for the production and accumulation of fructose polymers which would also be applicable to other polymers synthesized by proteins within the sucrase family of enzymes. The methods described may be used in alternate agronomic crops that accumulate significant quantities of sucrose, such as sugar beet and sugarcane. In addition fructan containing crops, such as chicory or Jerusalem artichoke may be improved by the methods described in the present invention. The effect of this technology is a method of large scale production of unique polymers, especially fructans, at reduced cost which have use as sweeteners, a polymer with beneficial properties for human-health (Hidaka and Hirayama, Biochem. Soc. Trans., 19:561-565 (1991)), probiotics in the animal feed industry (Hashimoto et al., U.S. Pat. No. 4,734,402 (1988); Nakamura et al., U.S. Pat. No. 4,788,065 (1988)), and may be used as chemical feedstock in new markets that would not otherwise be economically successful (Fuchs, Starke, 39:335-342, (1981); Fuchs, Biochem. Soc. Trans., 19:555-560, (1991)). Accumulation of fructans has been demonstrated not to be harmful to the growth, development or reproductive capacity of the transgenic plants.